Role of CXCR3 and CCR5 in allograft rejection

Chemokines are known to participate in allograft rejection by mediating leukocyte trafficking. Despite redundancy in chemokine family, several chemokine-chemokine receptor interactions have proven critical in alloimmune responses. We sought to determine the effect of combined blockade of CXCR3 and CCR5, two critical chemokine receptors, in acute rejection. METHODS: Heterotopic heart transplantation was performed using BALB/c to B6/129 mice deficient in CCR5. Following transplantation these mice were treated with goat anti-CXCR3 serum every other day. In the control group, BALB/c hearts were transplanted in wild type B6/129 recipients and treated with goat serum alone. No immunosuppression was given to either group. Recipient mice were then assessed daily for allograft function by abdominal palpation, and graft survival was confirmed by laparotomy. RESULTS: The donor hearts in the control group were rejected at 6 +/- 1 days posttransplantation. Combined blockade of CXCR3 and CCR5 prolonged allograft survival versus control; all allografts survived to 24 days. In addition, there was a decrease in graft infiltrating CD4 and CD8 lymphocytes in the experimental group at 24 days. CONCLUSION: Combined CXCR3 and CCR5 blockade is effective in prolonging allograft survival in a fully MHC mismatched murine model. Combined chemokine blockade holds promise in control of acute rejection in organ transplantation.     

CXCR3 and CCR5 positive T-cell recruitment in acute human renal allograft rejection

BACKGROUND: Experimental studies suggest that the infiltration of activated T cells into the allograft, the key event in the development of acute renal allograft rejection, depends on the expression of chemokines and their interaction with chemokine receptors expressed on T cells. METHODS: For a more detailed comprehension of the pathogenesis of T-cell recruitment in human acute rejection, the in situ expression of chemokines and chemokine receptors in allografts of 26 patients between day 3 and 9 after renal transplantation was examined in the present prospective study. RESULTS: Immunohistochemical staining showed a significantly increased number of CXCR3 (P<0.01) and CCR5 positive T cells (P<0.01) in the tubulointerstitium of patients with acute allograft rejection according to Banff grade Ia-IIb. Likewise the intrarenal RNA expression of the CXCR3 ligands IP-10 (5.2-fold) and I-TAC (7.2-fold) and the CCR5 ligand RANTES (5.7-fold), was significantly up-regulated in rejecting organs. In situ hybridization revealed that IP-10 but not I-TAC was predominantly expressed by infiltrating leukocytes in the tubulointerstitial area, co-localizing with CXCR3 positive T cells. To a lesser degree expression by tubular cells could be detected, providing a possible explanation for the increased urinary IP-10 excretion we found in patients with rejecting organs. CONCLUSIONS: These data from a prospective, biopsy-controlled study indicate that the local expression of IP-10 and RANTES leads to the directional movement of activated CXCR3 and CCR5 bearing T cells into the renal allograft and mediates acute rejection. Our data provide a rationale that blocking CXCR3 and CCR5 may offer a unique therapeutic approach to prevent episodes of acute rejection in the early phase after renal transplantation.     

Beneficial effects of targeting CCR5 in allograft recipients

BACKGROUND: The chemokine receptor, CCR5, and its three high-affinity ligands, macrophage inflammatory protein- (MIP) 1alpha, MIP-1beta, and regulated on activation normal T cell expressed and secreted (RANTES), are expressed by infiltrating mononuclear cells during the rejection of clinical and experimental organ allografts, although the significance of these molecules in the pathogenesis of rejection has not been established. METHODS: We studied intragraft events in four allograft models. First, we studied cardiac transplants in fully MHC-mismatched mice that were deficient in CCR5 or two of its ligands, MIP-1alpha or RANTES. Second we tested the effects of a neutralizing rat anti-mCCR5 monoclonal antibody on allograft survival. Third we assessed whether a subtherapeutic course of cyclosporine would potentiate enhance survival in CCR5-deficient recipients. Finally, we tested the effect of targeting CCR5 in a class II-mismatched model. RESULTS: Whereas mice deficient in expression of MIP-1alpha or RANTES reject fully MHC-mismatched cardiac allografts normally, CCR5-/- mice, or CCR5+/+ mice treated with a neutralizing mAb to mCCR5, show enhanced allograft survival. MHC class II-disparate mismatched are permanently accepted in CCR5-/- but not CCR5+/+ recipients. Finally, the beneficial effects of targeting of CCR5 are markedly synergistic with the effects of cyclosporine, resulting in permanent engraftment without development of chronic rejection. CONCLUSIONS: We conclude that CCR5 plays a key role in the mechanisms of host T cell and macrophage recruitment and allograft rejection, such that targeting of CCR5 clinically may be of therapeutic significance.     

A novel CCR5/CXCR3 antagonist protects intestinal ischemia/reperfusion injury

Chemokines and chemokine receptors have been demonstrated to be critical regulators in a variety of physiologic and pathologic immune responses. In particular, CCR5 and CXCR3 have been reported to play important roles in the alloimmune response. In this study, we investigated the therapeutic efficacy of a novel small-molecule compound, TAK779, an antagonist targeting both CCR5 and CXCR3 in intestinal ischemia/reperfusion (I/R) injury. We utilized an established murine intestinal I/R injury model. TAK779 treatment significantly improved mouse survival after 60 minutes of intestinal ischemia. We then examined the local intestinal expression of several cytokines and chemokines at 2 hours after reperfusion using real-time PCR. TAK779 treatment downregulated the expression of several cytokines, including TNF-alpha, IFN-gamma, and IL-4, suggesting that the beneficial effect of TAK779 was associated with inhibition of local immune activation. We further examined the systemic response after TAK779 treatment. Lung tissue damage was significantly prevented by the treatment, as determined by lung wet-to-dry weight ratios at 4 hours after intestinal I/R injury. In addition, we observed that CCR5 expression in the lung was significantly downregulated by the treatment, suggesting that TAK779 inhibited the infiltration of CCR5-positive cells into the remote organ. Our data suggest the critical role of CCR5 and CXCR3 in intestinal I/R injury and therapeutic efficacy of a novel small compound, TAK779, for protection against the intestinal I/R injury.     

A novel CD154 monoclonal antibody in acute and chronic rat vascularized cardiac allograft rejection

BACKGROUND: The CD40-CD154 interaction is critically important in the cell-mediated immune responses. Blockade of this costimulatory pathway has been shown to prevent acute allograft rejection in murine, as well as nonhuman primate models. However, the role of the CD40-CD154 pathway in the development of chronic rejection and the effects of CD154 targeting on progression of chronic rejection have not been evaluated. METHODS: We examined the effect of AH.F5, a new hamster anti-rat CD154 monoclonal antibody, in a fully allogeneic acute(u) into Lewis [LEW] (RT11) and chronic [WF.1L (RT1l) into LEW (RT1l)] vascularized cardiac allograft rejection model. In the chronic model, the antibody was evaluated for prevention (starting day of transplant) and interruption of progression (starting day 30 or 60 after transplant) of chronic vasculopathy. Graft survival, morphology, and immunohistology were evaluated. RESULTS: In the acute rejection model, anti-CD154 therapy alone prevented acute allograft rejection and resulted in 50% long-term allograft survival (>200 days) and donor-specific tolerance. In recipients treated with anti-CD154 monoclonal antibody in combination with a short course of cyclosporine, 100% of allografts survived long-term and all recipients achieved donor-specific tolerance. In the chronic rejection model, allografts from animals treated with the anti-CD154 antibody had a statistically significant lower score of graft arteriosclerosis and fibrosis in both the prevention and 30-day interruption groups when compared with control allografts. In addition, immunohistochemistry showed a decrease in intragraft mononuclear cell infiltration and activation. CONCLUSION: A new anti-CD154 antibody not only prevents acute allograft rejection, but also inhibits and interrupts the development of chronic rejection. In the acute rejection model cyclosporine acts synergistically with anti-CD154 therapy to prolong allograft survival and induce tolerance. In the chronic rejection model relatively early initiation of therapy is essential to prevent progression of chronic allograft vasculopathy and fibrosis.     

Differential expression of beta-chemokines MCP-1 and RANTES and their receptors CCR1, CCR2, CCR5 in acute rejection and chronic allograft nephropathy of human renal allografts

BACKGROUND: The beta-chemokines MCP-1 (CCL2) and RANTES (CCL5) have been shown to play important roles in acute renal transplant rejection (AR) and chronic allograft nephropathy (CAN). The potential relationship of expression of these chemokines, their chemokine receptors CCR1, CCR2, CCR5, and the cell populations of inflammatory infiltrate, histological and clinical diagnoses were investigated in biopsies at the time of AR and compared with biopsies of CAN. METHODS: In 24 renal transplant biopsies with AR (n = 15) and CAN (n = 9), the expression of MCP-1 and RANTES, their receptors CCR1, CCR2, and CCR5 and the infiltration with monocytes/macrophages and T cells were studied. RESULTS: As previously described, chemokine and chemokine receptor expression was found mainly in mononuclear cells infiltrating the interstitium and glomeruli. In the tubulointerstitial area and glomeruli the expression of MCP-1, RANTES, and their receptors correlated with an infiltration by monocytes/macrophages. Biopsies with CAN revealed a lower expression of MCP-1, RANTES, CCR1, CCR2 and CCR5 in tubulointerstitial cells, and a significantly lower infiltration with MRP14-positive monocytes/macrophages than biopsies with AR. In AR, MCP-1 and CCR1 showed a lower expression compared to RANTES, CCR2, and CCR5. CONCLUSIONS: The positive correlation between chemokines and chemokine receptors and infiltrating leukocytes during acute rejection, the lower but detectable expression of MCP-1, RANTES, CCR1, CCR2 and CCR5 in CAN, and the differences in the quantity of expression between the different chemokines and chemokine receptors point to a complex regulation of chemokine expression in renal allografts. Since chemokines are not only involved in inflammation but also in tissue regeneration, this could have impact on the development of CAN.     

Mechanisms of targeting CD28 by a signaling monoclonal antibody in acute and chronic allograft rejection

There is increasing evidence that ongoing T-cell recognition of alloantigen and activation are key mediators of chronic allograft rejection. The CD28-B7 pathway is unique among costimulatory pathways in that two alternate ligands for B7 exist: CD28 and CTLA4. Recently, it has been suggested that CTLA4 negative signaling may be required for induction of acquired tolerance in vivo. A strategy by which the T cell is targeted at the CD28 receptor rather than its ligands would theoretically allow the inhibitory functions of the CTLA4-B7-1/2 axis to remain intact. Using a rat-specific monoclonal antibody, we investigated the effect of targeting CD28 in a model of chronic rejection without the confounding variable of immunosuppression. We also used an acute cardiac allograft rejection model to investigate CD28 stimulation-based strategies to induce donor-specific tolerance. We demonstrated that anti-CD28 monoclonal antibody was as effective as CTLA4 immunoglobulin in protecting against chronic allograft vasculopathy. In addition, a short course of cyclosporine therapy synergized with either anti-CD28 monoclonal antibody or CTLA4 immunoglobulin, suggesting that it may be clinically relevant to combine low-dose calcineurin inhibitors with CTLA4 immunoglobulin or anti-B7 antibodies. Finally, we report on the potential mechanisms of action of targeting CD28 in vivo.     

抑制趋化因子受体CCR5减轻小鼠同种异体移植心脏急性排斥反应的研究

目的 研究抑制趋化因子受体CCR5减轻小鼠同种异体移植心脏急性排斥反应的作用机制.方法 采用小鼠颈部心脏移植模型,将96只小鼠用随机数字表法分为4组,每组24只,供、受者各12只,A组术后给予anti-CCR5 mAb和CsA,B组术后给予anti-CCR5 mAb,C组术后给予CsA,D组为对照组,术后给予生理盐水.于术后第7d取各组移植心组织6例,检测CCR5及IL-2和IL-10的表达差异,其余6例用于观察移植心脏存活时间.结果 A、B、C组小鼠移植心脏存活时间明显延长,其CCR5及IL-2的表达较D组明显减少,IL-10的表达明显增加.结论 抑制趋化因子受体CCR5对同种异体移植心脏有明显的保护作用,可能与细胞因子的表达有关.     

A proteasome inhibitor effectively prevents mouse heart allograft rejection

BACKGROUND: We have previously demonstrated in vitro that proteasome inhibitors could suppress proliferation and induce apoptosis of activated T cells. This finding suggests that such inhibitors could be used as a novel category of immunosuppressants in blocking allograft rejection. METHODS: The proteasome inhibitor dipeptide boronic acid (DPBA) was tested in vitro for its inhibitory effect on mouse T-cell proliferation and lymphokine secretion. DPBA was also used in vivo to treat mouse heterotopic heart allograft rejection. Possible side effects of this compound were examined according to blood chemistry of mice treated with DPBA. RESULTS: DPBA suppressed the T-cell proliferation and potently inhibited interleukin (IL)-2, IL-6, IL-10, IL-13, and IFN-gamma produced by anti-CD3-activated T cells. Given i.p. starting 1 day after transplantation at 0.66 mg/kg per day for 16 days, or at 1 mg/kg per day for 4 days followed by 0.5 mg/kg per day for 12 days, DPBA could prolong heart allograft survival to 35.5 days (mean survival time, MST) and to 36.2 days, respectively. The control group had MST of 7.3 days. When administrated 72 hr post operation at 1 mg/kg per day for 4 days, DPBA could prolong the graft survival to 19.8 days. During the course of these effective dosages, DPBA had no apparent toxicity in the liver, kidney, pancreas, or heart, according to analysis of blood chemistry. CONCLUSIONS: The proteasome inhibitor could repress allograft rejection in mice without apparent side-effects at the effective dosages. This finding has opened a new dimension in development of novel immunosuppressants for organ transplantation.     

A novel pathway of chronic allograft rejection mediated by NK cells and alloantibody

Chronic allograft vasculopathy (CAV) in murine heart allografts can be elicited by adoptive transfer of donor specific antibody (DSA) to class I MHC antigens and is independent of complement. Here we address the mechanism by which DSA causes CAV. B6.RAG1^?/? or B6.RAG1^?/?C3^?/? (H‐2^b) mice received B10.BR (H‐2^k) heart allografts and repeated doses of IgG2a, IgG1 or F(ab’)₂ fragments of IgG2a DSA (anti‐H‐2^k). Intact DSA regularly elicited markedly stenotic CAV in recipients over 28 days. In contrast, depletion of NK cells with anti‐NK1.1 reduced significantly DSA‐induced CAV, as judged morphometrically. Recipients genetically deficient in mature NK cells (γ‐chain knock out) also showed decreased severity of DSA‐induced CAV. Direct NK reactivity to the graft was not necessary. F(ab’)₂ DSA fragments, even at doses twofold higher than intact DSA, were inactive. Graft microvascular endothelial cells responded to DSA in vivo by increased expression of phospho‐extracellular signal‐regulated kinase (pERK), a response not elicited by F(ab’)₂ DSA. We conclude that antibody mediates CAV through NK cells, by an Fc dependent manner. This new pathway adds to the possible mechanisms of chronic rejection and may relate to the recently described C4d‐negative chronic antibody‐mediated rejection in humans.     

The role of CC chemokine receptor 5 (CCR5) in islet allograft rejection

Chemokines are important regulators in the development, differentiation, and anatomic location of leukocytes. CC chemokine receptor 5 (CCR5) is expressed preferentially by CD4(+) T helper 1 (Th1) cells. We sought to determine the role of CCR5 in islet allograft rejection in a streptozotocin-induced diabetic mouse model. BALB/c islet allografts transplanted into CCR5(-/-) (C57BL/6) recipients survived significantly longer (mean survival time, 38 +/- 8 days) compared with those transplanted into wild-type control mice (10 +/- 2 days; P < 0.0001). Twenty percent of islet allografts in CCR5(-/-) animals without other treatment survived >90 days. In CCR5(-/-) mice, intragraft mRNA expression of interleukin-4 and -5 was increased, whereas that of interferon-gamma was decreased, corresponding to a Th2 pattern of T-cell activation in the target tissues compared with a Th1 pattern observed in controls. A similar Th2 response pattern was also observed in the periphery (splenocytes responding to donor cells) by enzyme-linked immunosorbent spot assay. We conclude that CCR5 plays an important role in orchestrating the Th1 immune response leading to islet allograft rejection. Targeting this chemokine receptor, therefore, may provide a clinically useful strategy to prevent islet allograft rejection.     

Role of CXCR3 in cellular but not humoral renal allograft rejection

CXCR3, a chemokine receptor mainly expressed by T cells, is involved in animal transplant models and in human allograft rejection. CXCR3 expression was localized in formalin-fixed, paraffin-embedded renal allograft biopsies without signs of rejection (C4d-negative, Banff 0, n = 16), with C4d deposits as a sign of humoral rejection (C4d-positive, Banff 0, n = 8), with cellular rejection (C4d-negative, Banff I, n = 7) and with signs of both cellular and humoral rejection (C4d-positive, Banff 1, n = 5). Small, round infiltrating cells were CXCR3-positive. A high number of these cells was present in biopsies with cellular rejection (independent of C4d deposition). CXCR3-positive cells diffusely infiltrated the interstitium, including the tubular epithelium (tubulitis). CXCR3 scores and the area of CXCR3 staining were significantly higher in cellular rejection, when compared to biopsies without rejection, and with deposition of C4d alone. CXCR3-positive cells infiltrate renal allografts during cellular rejection, whereas C4d deposition is not associated with the recruitment of these cells.